Retrotransposable elements are abundant in all eukaryotic genomes. These elements have undoubtly played a significant role in reshaping these genomes, can account for many insertional mutations and chromosomal rearrangements, and are believed to be the origin of several viruses, most significantly retroviruses. Those retrotransposable elements with long-terminal repeats (LTRs) are well-studied and are believed to integrate into the genome by mechanisms similar to retroviruses. A second class of retrotransposable elements, termed here the non-LTR elements, have more recently been discovered and appear to have a fundamentally different mechanism for integration. The specific aim of this proposal is to study the mechanism of retrotransposition of the non-LTR element, R2. This element inserts specifically in the 28S genes of a wide variety of insects. It is known that the R2 element encodes an endonuclease that requires RNA before it can specifically cleave the 28S gene insertion site. The same protein also has reverse transcriptase activity. The specific integration of this element and the ability to express the entire coding capacity of this element in E. coli makes it ideally suited to test basic features of current models of non-LTR retrotransposition. Comparative studies will be conducted with the R2 element of both Bombyx mori and Drosophila melanogaster. The critical questions to be addressed are (a) whether the R2 protein is capable of specifically binding the R2 RNA transcript, and if so what sequences, (b) the nature of the interaction between the endonuclease with the DNA target site before and after cleavage, and (c) whether the cleaved DNA, which is not complimentary to the R2 element, can serve as a primer for reverse transcription. We will also attempt to obtain complete integration of an R2 element into a ribosomal DNA repeat in vivo by injecting the R2-- transcript/R2 protein complex into tissue culture cells. We will use a highly sensitive (PCR) assay to screen for integration. Finally, to lay the foundation for our long term goal of understanding the developmental regulation of R2 retrotransposition, we will attempt to find in D. melanogaster genetic conditions underwhich R2 elements are transcribed and translated, and whether this correlates with the integration of new R2 copies.